Domain Swapping in Abiotic Foldamers.

Foldamer sequences that adopt tertiary helix-turn-helix folds mediated by helix-helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain-swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

Enhancing the Features of DNA Mimic Foldamers for Structural Investigations.

DNA mimic foldamers based on aromatic oligoamide helices bearing anionic phosphonate side chains have been shown to bind to DNA-binding proteins sometimes orders of magnitude better than DNA itself. Here, we introduce new features in the DNA mimic foldamers to facilitate structural investigations of their interactions with proteins. Thirteen new foldamer sequences have been synthesized and characterized using NMR, circular dichroism, molecular modeling, and X-ray crystallography. The results show that foldamer helix handedness can be quantitatively biased by means of a single stereogenic center, that the foldamer structure can be made C2-symmetrical as in palindromic B-DNA sequences, and that associations between foldamer helices can be promoted utilizing dedicated C-terminal residues that act as sticky ends in B-DNA structures.

Molecular Sensing and Manipulation of Protein Oligomerization in Membrane Nanotubes with Bolaamphiphilic Foldamers.

Adaptive and reversible self-assembly of supramolecular protein structures is a fundamental characteristic of dynamic living matter. However, the quantitative detection and assessment of the emergence of mesoscale protein complexes from small and dynamic oligomeric precursors remains highly challenging. Here, we present a novel approach utilizing a short membrane nanotube (sNT) pulled from a planar membrane reservoir as nanotemplates for molecular reconstruction, manipulation, and sensing of protein oligomerization and self-assembly at the mesoscale. The sNT reports changes in membrane shape and rigidity caused by membrane-bound proteins as variations of the ionic conductivity of the sNT lumen. To confine oligomerization to the sNT, we have designed and synthesized rigid oligoamide foldamer tapes (ROFTs). Charged ROFTs incorporate into the planar and sNT membranes, mediate protein binding to the membranes, and, driven by the luminal electric field, shuttle the bound proteins between the sNT and planar membranes. Using Annexin-V (AnV) as a prototype, we show that the sNT detects AnV oligomers shuttled into the nanotube by ROFTs. Accumulation of AnV on the sNT induces its self-assembly into a curved lattice, restricting the sNT geometry and inhibiting the material uptake from the reservoir during the sNT extension, leading to the sNT fission. By comparing the spontaneous and ROFT-mediated entry of AnV into the sNT, we reveal how intricate membrane curvature sensing by small AnV oligomers controls the lattice self-assembly. These results establish sNT-ROFT as a powerful tool for molecular reconstruction and functional analyses of protein oligomerization and self-assembly, with broad application to various membrane processes.

Controlling aromatic helix dimerization in water by tuning charge repulsions.

Several helically folded aromatic oligoamides were designed and synthesized. The sequences were all water-soluble thanks to the charged side chains borne by the monomers. Replacing a few, sometimes only two, charged side chains by neutral methoxy groups was shown to trigger the formation of various aggregates which could be tentatively assigned to head-to-head stacked dimers of single helices, double helical duplexes and a quadruplex, none of which would form in organic solvent with organic-soluble analogues. The nature of the aggregates was supported by concentration and solvent dependent NMR studies, 1H DOSY experiments, mass spectrometry, and X-ray crystallography or energy-minimized models, as well as analogies with earlier studies. The hydrophobic effect appears to be the main driving force for aggregation but it can be finely modulated by the presence or absence of a small number of charges to an extent that had no precedent in aromatic foldamer architectures. These results will serve as a benchmark for future foldamer design in water.

High-Affinity Hybridization of Complementary Aromatic Oligoamide Strands in Water.

We prepared a series of water-soluble aromatic oligoamide sequences all composed of a segment prone to form a single helix and a segment prone to dimerize into a double helix. These sequences exclusively assemble as antiparallel duplexes. The modification of the duplex inner rim by varying the nature of the substituents borne by the aromatic monomers allowed us to identify sequences that can hybridize by combining two chemically different strands, with high affinity and complete selectivity in water. X-ray crystallography confirmed the expected antiparallel configuration of the duplexes whereas NMR spectroscopy and mass spectrometry allowed us to assess precisely the extent of the hybridization. The hybridization kinetics of the aromatic strands was shown to depend on both the nature of the substituents responsible for strand complementarity and the length of the aromatic strand. These results highlight the great potential of aromatic hetero-duplex as a tool to construct non-symmetrical dynamic supramolecular assemblies.

Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

DNA mimic foldamers affect chromatin composition and disturb cell cycle progression.

The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Self-assembly of achiral building blocks into chiral cyclophanes using non-directional interactions.

The diastereoselective assembly of achiral constituents through a single spontaneous process into complex covalent architectures bearing multiple stereogenic elements still remains a challenge for synthetic chemists. Here, we show that such an extreme level of control can be achieved by implementing stereo-electronic information on synthetic organic building blocks and templates and that non-directional interactions (i.e., electrostatic and steric interactions) can transfer this information to deliver, after self-assembly, high-molecular weight macrocyclic species carrying up to 16 stereogenic elements. Beyond the field of supramolecular chemistry, this proof of concept should stimulate the on-demand production of highly structured polyfunctional architectures.

High-affinity single and double helical pseudofoldaxanes with cationic guests.

Two aromatic oligoamides, the 8-residue H8 and 16-residue H16, that adopt stable, cavity-containing helical conformations were examined for their complexation of a rodlike dicationic guest, octyl viologen (OV2+) and para-bis(trimethylammonium)benzene (TB2+). Studies based on 1D and 2D 1H NMR, isothermal titration calorimetry (ITC), and X-ray crystallography demonstrated that H8 and H16 wraps around two OV2+ ions as a double helix and a single helix, respectively, resulting in 2 : 2 and 1 : 2 complexes. Compared to H8, the longer H16 binds the OV2+ ions with much higher binding affinity and with extraordinary negative cooperativity. In contrast to its 1 : 2 binding with OV2+, the binding of helix H16 with the bulkier guest TB2+ shows a 1 : 1 ratio. Host H16 also selectively binds OV2+ in the presence of TB2+. This novel host-guest system features pairwise placement of the otherwise strongly repulsive OV2+ ions in the same cavity, strong negative cooperativity, and mutual adaptability of hosts and guests. The resultant complexes are highly stable [2]-, [3]-, and [4]pseudo-foldaxanes with few known precedents.

An abiotic, tetrameric, eight-helix bundle.

Four helically folded aromatic oligoamide sequences containing either a chiral monomer based on 2-(2-aminophenoxy)-propionic acid, an N-terminal (1H)-camphanyl group, or both, were synthesized. Spectroscopic solution investigations using 1H NMR and circular dichroism (CD) demonstrated that the 2-(2-aminophenoxy)-propionic acid unit biases helix handedness quantitatively in chloroform and dichloromethane. It even quantitatively overcomes an opposing effect of the camphanyl group and thus ensures reliable helix handedness control. A series of nine sequences composed of two helically folded aromatic oligoamide segments separated by a flexible linker based on a di-, tri- or tetraethylene glycol unit were then synthesized. In these sequences, helix handedness was controlled by means of an N-terminal (1H)-camphanyl group or a 2-(2-aminophenoxy)-propionic acid units in either both helical segments, or only in the N-terminal segment, or in none of the segments. The helical segments all displayed hydroxy and carbonyl groups at their surfaces as hydrogen bond donors and acceptors so as to promote helix-to-helix hydrogen bonding. NMR and CD spectroscopic studies showed that, in some cases, well-defined, stable, discrete abiotic helix-turn-helix tertiary folds form in organic solvents. Molecular modelling suggests that these correspond to structures in which the two helix axes are at an angle. In one case, the absence of handedness control resulted in a complex and large aggregate. A solid state structure obtained by single crystal X-ray diffraction analysis revealed a tetrameric assembly composed of eight helices with both right and left handedness arranged in three subdomains consisting of two hydrogen-bonded three-helix bundles and one two-helix-bundle. Several helix-to-helix hydrogen bonds were mediated by bridging water molecules. This structure constitutes an important milestone in the construction of abiotic protein-like architectures.

Coaxial assembly of helical aromatic foldamers by metal coordination.

Deprotonation of acid-terminated helical aromatic foldamers with a mineral base in chlorinated solvents led to their dimerization through the coordination of a metal cation (Li+, Na+, K+, Ag+, or Hg2+) with the terminal carboxylate functions. This new ligation method was applied to oligomerize diacid-functionalized foldamers.

Optimization and Automation of Helical Aromatic Oligoamide Foldamer Solid-Phase Synthesis.

Helically folded oligoamides of 8-amino-2-quinolinecarboxylic acid composed of up to 41 units were prepared using optimized manual solid-phase synthesis (SPS). The high yield and purity of the final products places these SPS protocols among the most efficient known to date. Furthermore, analytical methods allowing for the clear identification and purity assessment of the products were validated, including 1 H NMR, a seldom used method for such large molecules. Adaption of the SPS protocols, in particular using in situ acid chloride activation under Appel's conditions, made it possible to efficiently implement SPS on a commercial peptide synthesizer, leading to a dramatic reduction of the laboratory work required to produce long sequences. Automation constitutes a breakthrough for the development of helical aromatic oligoamide foldamers.

Combining local conformational preferences and solvophobic effects in helical aromatic oligoamide foldamers.

Aromatic oligoamide foldamers were designed using a newly-developed monomer so that helical folding was promoted by both local conformation preferences and solvophobic effects. Solid phase synthesis provided quick access to the desired sequences. Sharp solvent-driven conformational transitions that depended on sequence length were evidenced by both NMR and UV absorption spectroscopies.

Molecular torsion springs: alteration of helix curvature in frustrated tertiary folds.

The first abiotic foldamer tertiary structures have been recently reported in the form of aromatic helix-turn-helix motifs based on oligo-quinolinecarboxamides held together by intramolecular hydrogen bonds. Tertiary folds were predicted by computational modelling of the hydrogen-bonding interfaces between helices and later verified by X-ray crystallography. However, the prognosis of how the conformational preference inherent to each helix influences the tertiary structure warranted further investigation. Several new helix-turn-helix sequences were synthesised in which some hydrogen bonds have been removed. Contrary to expectations, this change did not strongly destabilise the tertiary folds. On closer inspection, a new crystal structure revealed that helices adopt their natural curvature when some hydrogen bonds are missing and undergo some spring torsion upon forming the said hydrogen bonds, thus potentially giving rise to a conformational frustration. This phenomenon sheds light on the aggregation behaviour of the helices when they are not linked by a turn unit.

Homochiral versus Heterochiral Dimeric Helical Foldamer Bundles: Chlorinated-Solvent-Dependent Self-Sorting.

Aromatic oligoamide sequences programmed to fold into stable helical conformations were designed to display a linear array of hydrogen-bond donors and acceptors at their surface. Sequences were prepared by solid-phase synthesis. Solution 1 H NMR spectroscopic studies and solid-state crystallographic structures demonstrated the formation of stable hydrogen-bond-mediated dimeric helix bundles that could be either heterochiral (with a P and an M helix) or homochiral (with two P or two M helices). Formation of the hetero- or homochiral dimers could be driven quantitatively using different chlorinated solvents-exemplifying a remarkable case of either social or narcissistic chiral self-sorting or upon imposing absolute handedness to the helices to forbid PM species.

(Re-)Directing Oligomerization of a Single Building Block into Two Specific Dynamic Covalent Foldamers through pH.

Dynamic foldamers are synthetic folded molecules which can change their conformation in response to an external stimulus and are currently at the forefront of foldamer chemistry. However, constitutionally dynamic foldamers, which can change not only their conformation but also their molecular constitution in response to their environment, are without precedent. We now report a size- and shape-switching small dynamic covalent foldamer network which responds to changes in pH. Specifically, acidic conditions direct the oligomerization of a dipeptide-based building block into a 16-subunit macrocycle with well-defined conformation and with high selectivity. At higher pH the same building block yields another cyclic foldamer with a smaller ring size (9mer). The two foldamers readily and repeatedly interconvert upon adjustment of the pH of the solution. We have previously shown that addition of a template can direct oligomerization of the same building block to yet other rings sizes (including a 12mer and a 13mer, accompanied by a minor amount of 14mer). This brings the total number of discrete foldamers that can be accessed from a single building block to five. For a single building block system to exhibit such highly diverse structure space is unique and sets this system of foldamers apart from proteins. Furthermore, the emergence of constitutional dynamicity opens up new avenues to foldamers with adaptive behavior.

Isotope Ratio Encoding of Sequence-Defined Oligomers.

Information storage at the molecular level commonly entails encoding in the form of ordered sequences of different monomers and subsequent fragmentation and tandem mass spectrometry analysis to read this information. Recent approaches also include the use of mixtures of distinct molecules noncovalently bonded to one another. Here, we present an alternate isotope ratio encoding approach utilizing deuterium-labeled monomers to produce hundreds of oligomers endowed with unique isotope distribution patterns. Mass spectrometric recognition of these patterns then allowed us to directly readout encoded information with high fidelity. Specifically, we show that all 256 tetramers composed of four different monomers of identical constitution can be distinguished by their mass fingerprint using mono-, di-, tri-, and tetradeuterated building blocks. The method is robust to experimental errors and does not require the most sophisticated mass spectrometry instrumentation. Such isotope ratio-encoded oligomers may serve as tags that carry information, but the method mainly opens up the capability to write information, for example, about molecular identity, directly into a pure compound via its isotopologue distribution obviating the need for additional tagging and avoiding the use of mixtures of different molecules.

Directing the Self-Assembly of Aromatic Foldamer Helices using Acridine Appendages and Metal Coordination.

Folded molecules provide complex interaction interfaces amenable to sophisticated self-assembly motifs. Because of their high conformational stability, aromatic foldamers constitute suitable candidates for the rational elaboration of self-assembled architectures. Several multiturn helical aromatic oligoamides have been synthesized that possess arrays of acridine appendages pointing in one or two directions. The acridine units were shown to direct self-assembly in the solid state via aromatic stacking leading to recurrent helix-helix association patterns under the form of discrete dimers or extended arrays. In the presence of Pd(II), metal coordination of the acridine units overwhelms other forces and generates new metal-mediated multihelical self-assemblies, including macrocycles. These observations demonstrate simple access to different types of foldamer-containing architectures, ranging from discrete objects to 1D and, by extension, 2D and 3D arrays.

Differential Peptide Multi-Macrocyclizations at the Surface of a Helical Foldamer Template.

Hybrid sequences comprising a peptide with several Cys residues and an aromatic foldamer helix with several chloroacetamide functions at its surface were synthesized. Such products may in principle form numerous macromulticyclic thioether products by intramolecularly combining all Cys residues and all chloroacetamide functions. However, we show that the reactive sites on the structurally defined helix can be placed at such locations that the peptide selectively stitches itself to form a series of different macrocycles within mostly one preferred product. Reactions were monitored by HPLC and products with two, three or four macrocycles were identified using LC-MS and NMR. The series of selective macrocyclizations define a sort of reaction trail where reaction sites otherwise identical are involved successively because of their precise positioning in space. The trails can be predicted to a large extent based on structural considerations and the assumption that smaller macrocycles form faster.

[3]Foldarotaxane-mediated synthesis of an improbable [2]rotaxane.

The wrapping of an aromatic oligoamide helix around an active ester-containing [2]rotaxane enforced the sliding and the sequestration of the surrounding macrocycle around a part of the axle for which it has no formal affinity. The foldamer-mediated compartmentalization of the [2]rotaxane shuttle was subsequently used to prepare an improbable rotaxane.